Retinoic acid, a natural derivative of vitamin A, is essential for vertebrate growth, development and homeostasis. It, as well as a number of synthetic analogs are important therapeutic agents in the treatment of dermatologic diseases and in both chemoprevention and the treatment of certain cancers. However their use is limited by their concomitant toxicity (e.g. teratogenicity). Retinoic acid interacts with two classes of intracellular retinoic acid binding proteins; (i) specific nuclear receptors which belong to the steroid/thyroid hormone receptor superfamily of transcriptional regulators (RAR alpha, beta, tau and RXR alpha, beta, tau) and (ii) small cytoplasmic binding proteins that belong to a family of small intracellular lipid binding proteins (CRABP I and II). Retinoid signalling pathways involve a complex interplay between the various nuclear receptors and the cytoplasmic binding proteins. The RXRs serve as auxiliary proteins for a number of hormone signalling pathways in addition to retinoic acid. Compounds which exhibit more selectively binding properties may have more restricted biological effects that result in lower toxicity associated with clinical use. We propose to study the structural basis of ligand-protein interactions by expressing and purifying these binding proteins in E. coil. Isotope directed-nmr methods will be applied to directly observe the bound conformation of 13C-labeled retinoic acid and its contact points with neighboring amino acid side chains within each binding protein complex. NMR constraints will be incorporated in molecular modeling of the protein complex using available structural information, to generated hypotheses regarding the important determinants for molecular recognition. The solution structures of the two cytoplasmic proteins uniformly enriched by 15N and 13C, will be determined. The ligand protein interface will be modified by examining various synthetic analogs and protein mutants, to test hypotheses on the determinants important for molecular recognition. A systematic biophysical analysis of a series of purified truncated receptors will provide further insights on the interaction between ligand binding and receptor dimerization and DNA-binding.